Genes differentially expressed in breast cancer

ABSTRACT

A polynucleotide sequence as shown in SEQ ID NO:1 is associated with metastatic potential of cancer cells, especially breast cancer cells. Methods are provided for determining the risk of metastasis of a tumor, by determining whether a tissue sample from a tumor expresses a polypeptide or mRNA encoded by a polynucleotide as shown in SEQ ID NO:1. Also provided are therapeutic methods and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/758,575, filed Jan. 9, 2001, now pending, and claimspriority from U.S. Patent Application No. 60/175,462 filed Jan. 10,2000, which applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for predicting the behavior of tumors.More particularly, the invention relates to methods in which tumorsamples (primary and metastases) are examined for expression of aspecified gene.

2. Description of the Related Art

Breast cancer is one of the most common malignant diseases with about1,000,000 new cases per year worldwide. Despite use of a number ofhistochemical, genetic, and immunological markers, clinicians still havea difficult time predicting which tumors will metastasize to otherorgans. Some patients are in need of adjuvant therapy to preventrecurrence and metastasis and others are not. However, distinguishingbetween these subpopulations of patients is not straightforward, andcourse of treatment is not easily charted. There is a need in the artfor new markers for distinguishing between tumors which will or havemetastasized and those which are less likely to metastasize.

There is also a need in the art for markers of tumors, particularlymarkers that may also be found in metastatic tumors.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide markers fordetecting tumors, particularly those having a tendency to metastasize.These and other objects of the invention are provided by one or more ofthe embodiments described below.

One embodiment of the invention provides an isolated and purified humanprotein having an amino acid sequence which is at least 85% identical toan amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1or the complement thereof.

Another embodiment of the invention provides a fusion protein whichcomprises a first protein segment and a second protein segment fused toeach other by means of a peptide bond. The first protein segmentconsists of at least six contiguous amino acids selected from an aminoacid sequence encoded by a nucleotide sequence SEQ ID NO:1 or thecomplement thereof, and the second protein segment comprises an aminoacids sequence not found adjacent to the first protein segment in thenative protein encoded by SEQ ID NO:1.

Yet another embodiment of the invention provides an isolated andpurified polypeptide consisting of at least six contiguous amino acidsof a human protein having an amino acid sequence encoded by a nucleotidesequence of SEQ ID NO:1 or the complement thereof.

Still another embodiment of the invention provides a preparation ofantibodies which specifically bind to a human protein which comprises anamino acid sequence encoded by a nucleotide sequence of SEQ ID NO:1 orthe complement thereof.

Even another embodiment of the invention provides an isolated andpurified subgenomic polynucleotide comprising at least 11 contiguousnucleotides of a nucleotide sequence which is at least 95% identical toa nucleotide sequence of SEQ ID NO:1 or the complement thereof.

Another embodiment of the invention provides an isolated and purifiedpolynucleotide which comprises a coding sequence comprising a nucleotidesequence of SEQ ID NO:1 or the complement thereof.

Yet another embodiment of the invention provides a method foridentifying metastasis in a tissue sample. An expression product of agene which comprises a coding sequence of SEQ ID NO:1 is measured in anon-primary tumor tissue sample. A tissue sample which expresses theproduct at a higher level than in a control sample is categorized asbeing metastatic.

Yet a further embodiment of the invention provides a method fordetecting a human gene encoding SEQ ID NO:2, the method comprisingobtaining in computer-readable format SEQ ID NO:1, comparing thesequence with polynucleotide sequences of a human genome, andidentifying one or more human genome sequences having at least 95%sequence identity to SEQ ID NO:1 as determined by the Smith-Watermanalgorithm using an affine gap search with a gap open penalty of 12 and agap extension penalty of 1 as parameters.

The invention further provides a population of antibodies that can beused to detect breast cancer, wherein the antibodies are contacted withprimary breast cancer tissue, metastatic breast cancer tissue, and/or abody fluid of a person suspected of having breast cancer, therebydetecting a protein encoded by SEQ ID NO:1.

The invention also provides a kit for use in diagnosing breast cancer,comprising at least one ligand, such as an antibody, capable of bindingto a protein encoded by SEQ ID NO:1, wherein the ligand is detectablylabeled.

The invention thus provides the art with a number of polynucleotides andpolypeptides, which can be used as markers of metastasis. These areuseful for more rationally prescribing the course of therapy for breastcancer patients.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates the polynucleotide sequence of human Out at First(SEQ ID NO:1).

FIG. 2 illustrates the amino acid sequence encoded by SEQ ID NO:1 (SEQID NO:2).

FIG. 3 illustrates the putative signal peptide (SEQ ID NO:3).

FIG. 4 illustrates the translation of SEQ ID NO:1 (SEQ ID NO:1,polynucleotide; SEQ ID NO:2, amino acid sequence).

FIG. 5 illustrates the expression of hsOAF relative to β-Actin in tumorcell lines and tumor tissues from SCID mice developed from the celllines. “PT” refers to primary tumor.

FIG. 6 illustrates the growth of colonies by MDA-MB-435 cells in softagar following treatment with antisense oligo SEQ ID NO:4 (66-2as) orreverse control SEQ ID NO:5 (66-2rc), relative to untreated cells (WT).

FIG. 7 is an alignment of the human OAF amino acid sequence (SEQ ID NO:2) with the Drosophila OAF amino acid sequence (SEQ ID NO: 7).

FIG. 8. FIG. 8A illustrates the expression of hsOAF protein in COS-7 andMCF-7 cell lines. FIG. 8B illustrates the expression of hsOAF protein inmammory carcinoma cell lines.

FIG. 9 illustrates the expression of hsOAF in normal human tissues.

FIG. 10. FIG. 10A illustrates the morphological changes seen inMDA-MB-435 cells following treatment with antisense oligo (SEQ ID NO:4).AS=antisense; RC=reverse control (SEQ ID NO:5); M=conditioned medium.FIG. 10 B illustrates cell invasion following treatment of MDA-MB-435cells with AS, RC and RC+M.

FIG. 11 illustrates the predicted signal sequence of human OAF (SEQ IDNO: 9) (double underline), DNA sequence (SEQ ID NO:8).

FIG. 12. FIGS. 12A and 12B illustrate the secretion of hsOAF byMDA-MB-435 cells treated with antisense oligo (SEQ ID NO:4) or reversecontrol oligo (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE INVENTION

Metastasis of breast carcinomas and their proliferation at distant loci(lung and bone, mainly) is one of the more severe developments inpatients with breast cancer. Metastasis is a multistep process by whichtumor cells emigrate from the primary tumor, disseminate through bloodand lymph vessels, and then are deposited in specific target organswhere they re-colonize. Schirrmacher, V., Adv. Cancer Res. 43:1–73, 1985and Liotta, L. A. et al., Cell 64(2):327–36 (1991). During this processthe invasiveness of tumor cells is crucial since they must encounter andpass through numerous basement membranes. Liotta, L. A., Am. J. Pathol.117(3):339–48 (1984) and Fidler, I. J., Cancer Res. 38(9):2651–60(1978). Therefore the elucidation of the molecular causes of tumor cellinvasion and metastasis is essential for the development of efficienttreatment procedures for breast cancer patients. Genes expressed inbreast tumor metastasis are potential targets that play critical rolesduring metastasis. Identification of such genes and their biologicalfunction will significantly contribute to the development of therapy anddiagnosis for breast cancer.

Some important genes involved in breast tumor metastasis have beendiscovered. Loss of estrogen receptor and presence of vimentin have beenassociated with human breast tumor invasiveness and poor prognosis, andalso correlate with the invasiveness and metastatic potential of humanbreast cancer cell lines. Aamdal S., et al., Cancer 53(11):2525–9(1984); Clark, G. M., et al., Semin Oncol., 2 Suppl 1:20–5 (1988);Raymond, W. A. et al., J. Pathol. 157(4):299–306 (1989); Raymond, W. A.,et al., J. Pathol. 158(2):107–14 (1989); and Thompson, E. W. et al., J.Cell Physiol. 150(3):534–44 (1992). E-cadherin underexpression has beenimplicated in mammary tumor invasiveness. Vleminckx, K., et al., Cell66(1):107–19 (1991) and Oka, H., et al., Cancer Res. 53(7):1696–701(1993). Maspin, a protease inhibitor expressed in normal mammaryepithelial cells but not in most breast carcinoma cell lines, was ableto suppress MDA-MB-435 cells' ability to induce tumors and metastasizein mice and to invade basement membrane in vitro. Loss of maspinexpression occurred most frequently in advanced cancers. Zou, Z., etal., Science 263(5146):526–9 (1994) and Seftor, R. E., et al., CancerRes. 58(24):5681–5 (1998).

Overexpression of TIMP-4 (tissue inhibitor of metalloproteinases-4) orCLCA2 (Ca²⁺-activated chloride channel-2) in MDA-MB-435 cells bytransfection inhibited the tumorigenicity, invasiveness and metastasisability of the cells. Wang, M., et al., Oncogene 14(23):2767–74 (1997)and Gruber, A. D., et al., Cancer Res. 59(21):5488–91 (1999).Overexpression of the growth factor receptors IGF-IR and p185^(ErbB-2)has been found to be involved in breast cancer metastasis. Surmacz, E.,et al., Breast Cancer Res. Treat 47(3):255–67 (1998); Dunn, S. E., etal., Cancer Res. 58(15):3353–61 (1998); Tan, M., et al., Cancer Res.57(6):1199–205 (1997); Dhingra, K., et al., Semin Oncol. 23(4):436–45(1996); and Revillion, F., et al., Eur. J. Cancer 34(6):791–808 (1998).

The aspartyl protease cathepsin D has been reported to be a marker ofpoor prognosis for breast cancer patients and there is a significantcorrelation between high cathepsin D concentration in the cytosol ofprimary breast cancer and development of metastasis, though nocorrelation was found between cathepsin D secretion and invasion abilityof breast cancer cell lines. Rochefort, H., Breast Cancer Res Treat16(1):3–13 (1990); Johnson, M. D., et al., Cancer Res. 53(4):873–7(1993); and Rochefort, H., et al., Clin Chim Acta. 291(2):157–70 (2000).Osteopontin, a secreted integrin-binding glycoprotein that is thought tobe involved in bone resorption and bone formation, can induce migrationand invasion of mammary carcinoma cells. Osteopontin levels (tumor cellor plasma levels) have been associated with enhanced malignancy ofbreast cancer. Denhardt, D. T., et al., FASEB J. 7(15):1475–82 (1993);Denhardt, D. T., et al., J. Cell Biochem Suppl., 30–31:92–102 (1998);Tuck, A. B., et al., J. Cell Biochem. 78(3):465–75 (2000); Tuck, A. B.,et al., Oncogene 18(29):4237–46 (1999); and Singhal, H., et al., ClinCancer Res. 3(4):605–11 (1997).

The invention relates to the cloning of a novel gene first identified asbeing expressed in highly metastatic human breast cancer cell lines.Antibodies to the protein were raised and immunohistochemical stainingof breast tumor samples was performed. The protein was stronglyexpressed in 44/45 primary breast tumors, and in 26/26 metastasis. Thus,the protein is a marker for primary and metastatic breast cancer. It mayalso play a role particular to tumors with a tendency to metastasize.Because of its expression in primary and metastatic breast cancer, theprotein is useful in detecting such cancer in body fluids includingblood, which is consistent with the secretory nature of the protein.

The gene encodes a secreted protein and its protein secretion has beenconfirmed to be much greater in highly metastatic human breast cancercell lines than in low metastatic/nonmetastatic cell lines. Knockout ofthe secretion of this protein of the aggressive MDA-MB-435 cell line byantisense oligo technology resulted in significant morphologicalalteration along with reduced invasiveness and proliferation rate of thecells. The gene is named hsOAF based on its homology with the Drosophilagene OAF (out at first). Bergstrom, D. E., et al., Genetics139(3):1331–46 (1995) and Merli, C., et al., Genes Dev. 10(10):1260–70(1996).

This information can be utilized to make diagnostic reagents specificfor the expression products of the expressed gene. It can also be usedin diagnostic and prognostic methods which will help clinicians inplanning appropriate treatment regimes for cancers, especially of thebreast.

The polynucleotide is shown in FIG. 1 (SEQ ID NO:1), and the predictedopen reading frame (ORF) encodes a polypeptide shown in FIG. 2 (SEQ IDNO:2). The first 30 amino acid residues (SEQ ID NO:3) comprise aputative signal peptide, with a predicted protease cleavage siteindicated by “*”: APLLG * TGAPA (SEQ ID NO: 10) (between amino acids atpositions 25 and 26 of SEQ ID NO:3).

The polynucleotide sequence of the invention shares some homology with aDrosophila gene known as “Out at First” (oaf). Transcription of oafresults in three classes of alternatively polyadenylated RNAs, theexpression of which is developmentally regulated. All oaf transcriptscontain two adjacent ORFs separated by a single UGA stop codon.Suppression of the UGA codon during translation could lead to theproduction of different proteins from the same RNA molecule. Duringoogenesis, oaf RNA is expressed in nurse cells of all ages, and ismaternally contributed to the egg.

During embryonic development, zygotic transcription of the oaf geneoccurs in small clusters of cells in most or all segments at the time ofgermband extension and later in a segmentally repeated pattern in thedeveloping central nervous system. The oaf gene is also expressed in theembryonic, larval and adult gonads of both sexes. (Bergstrom, D. E. etal., Genetics 139:1331–1346, 1995.)

The polynucleotide of the invention was differentially expressed inseven pairs of high metastatic versus non-metastatic or low metastaticbreast cancer cell lines. The cell lines used are MDA-MB-361 (derivedfrom human breast adenocarcinoma), MDA-MB-231 (human breast cancer cellsmetastatic to bone and/or lung); MDA-MB-468 (derived from human estrogenreceptor-negative breast cancer cells); MCF-7 (non-metastatic humanbreast cancer cells); ZR-75-1 (derived from estrogen receptor-positivehuman breast carcinomas, Engle et al., Cancer Res. 38:3352–64 (1978));and MDA-MB-435 (derived from estrogen receptor-negative human breastcarcinoma cells, Rishi et al., Cancer Res. 56:5246–5252 (1996)).

The expression profile is as follows:

TABLE 1 Ratio of Cell Line Pair Expression MDA-MB-361/MDA-MB-231 0.11MDA-MB-468/MDA-MB-231 0.44 MCF-7/MDA-MB-231 0.17 ZR-75-1/MDA-MB-231 0.12MDA-MB-361/MDA-MB-435 0.06 MDA-MB-468/MDA-MB-435 0.36 MCF-7/MDA-MB-4350.03

The upregulation of the mRNA expression was confirmed by Northern blotanalysis using total RNA from the cell lines (FIG. 5).

The cell lines in which expression of the polynucleotide of theinvention was compared represent human breast cancers of varyingmetastatic potential. Cell line ZR-75-1 cultures were derived frommalignant ascitic effusion of a breast cancer patient. The cell linesgrown in vitro closely resembled the morphology seen in biopsies or cellpreparations from the donors of the original cells. ZR-75-1 cells arespecifically stimulated by estrogen, and have been used as a modelsystem for studying estrogen responsiveness. Engel, L. W. et al., CancerRes. 38:3352–3364, 1978.

Cell line MDA-MB-435 is an estrogen receptor-negative cell line that hasbeen studied as a model for human breast cancer, for example, forstudying the mechanism of action of growth inhibition in the presence ofretinoic acid. Rishi, A. K. et al., Cancer Res. 56:5246–5252, 1996.Growth inhibition by retinoids has also been studied in MCF-7 cells andMDA MB 468 cells. Tin-U, C. K. et al., Am. Soc. Clin. Onc. Proceedings,Vol. 17, 2125, 1998.

Cell line MDA-MB-361 was derived from a human breast adenocarcinoma,specifically from a malignant site. ATCC Number HTB-27. Differentialexpression of human Wnt genes has been studied in this cell line.Huguet, E. L. et al., Cancer Res. 54:2615–2621, 1994.

Once metastasis occurs, mammary primary tumor cells invade basementmembranes and spread to other organs of the body and the survival chanceof patients with breast cancer is reduced. It is critical to identifygenes participating in breast cancer invasion and metastasis on behalfof clinical diagnosis and therapy. Such genes are potential markers fordiagnosis or candidate targets for therapeutic drug development. Forinstance, presence of vimentin in human breast tumor has been associatedwith lack of estrogen receptor and tumor invasiveness as a marker ofpoor prognosis. Raymond, W. A. et al., J. Pathol. 157(4):299–306 (1989);Raymond, W. A., et al., J. Pathol. 158(2):107–14 (1989); and Thompson,E. W. et al., J. Cell Physiol. 150(3):534–44 (1992). Increasedactivities of matrix metalloproteinases are related with the metastaticphenotype of carcinomas, especially breast cancer. Basset, P., et al.,Nature 348(6303):699–704 (1990) and Basset, P., et al., Cancer 74(3Suppl):1045–9 (1994). Osteopontin, a secreted integrin-bindingglycoprotein, is able to induce increased invasiveness of human mammaryepithelial cells and has been associated with enhanced malignancy inbreast cancer. Tuck, A. B., et al., J. Cell Biochem. 78(3):465–75(2000); Tuck, A. B., et al., Oncogene 18(29):4237–46 (1999); andSinghal, H., et al., Clin Cancer Res. 3(4):605–11 (1997).

The invention relates to identification of a novel secreted protein(hsOAF) expressed in primary breast cancer and related metastasis. Thehuman breast cancer cell lines used in elucidating the role of theprotein are divided into three groups according to their metastaticabilities: highly metastatic, low metastatic, and nonmetastatic. Takingadvantage of different metastatic potentials among these cell linegroups and utilizing the advanced microarray technology, genes wereidentified which are differentially expressed between highly metastatichuman breast cancer cell lines and low metastatic/nonmetastatic ones.hsOAF gene is the focus of this invention as it encodes a novel secretedprotein, and is expressed in breast cancer tissue and metastatic breastcancer tissue.

To investigate the potential role of secreted hsOAF protein in breastcancer metastasis, antisense oligo technology was used to specificallyknock out hsOAF expression. Antisense oligo technology is an efficient,fast way to dramatically reduce gene expression for gene functionalstudies. Stein, C. A., et al., Science 261(5124):1004–12 (1993);Defacque, H. et al., J. Cell Physiol. 178(1):109–19 (1999). Knockout ofhsOAF protein secretion of highly metastatic MDA-MB-435 cells resultedin cell shape change, reduced cell invasiveness and slower cellproliferation. Treatment of cells with the conditioned medium (culturemedium of normal MDA-MB-435 cells) led to recovery of all thosephenotypic alterations caused by the knockout of hsOAF protein secretionto some degree. Although the inventors are not bound by a specificmechanism, the secreted hsOAF protein is believed to be involved in theinvasiveness and proliferation of MDA-MB-435 cells. However, knockout ofhsOAF protein secretion of another highly metastatic cell line,MDA-MB-231, by antisense oligo technology did not cause any significantcellular changes. MDA-MB-435 and MDA-MB-231 are quite differentmetastatic cell lines and MDA-MB-435 shows much stronger hsOAF proteinsecretion than does MDA-MB-231.

hsOAF gene is located at chromosome 11q23 region where loss ofheterozygosity occurs frequently in human breast tumors. Negrini, M., etal., Cancer Res 55(14):3003–7 (1995) and Tomlinson, I. P., et al., J.Clin. Pathol. 48(5):424–8 (1995). Loss of heterozygosity at 11q23 inprimary human breast tumors has been reported to be associated with poorsurvival after metastasis. Winqvist, R., et al., Cancer Res.55(12):2660–4 (1995). 11q23 also contains loci such as ATM(Ataxia-telangiectasia, mutated), and MLL (which is frequently disruptedby chromosomal rearrangement in acute leukemia). Rasio, D., et al.,Cancer Res. 55(24):6053–7 (1995) and Rubnitz, J. E., et al., Leukemia10(1):74–82 (1996). The relationship between mutation at chromosome11q23 and hsOAF gene expression in breast cancer metastasis remainsunclear.

Secreted hsOAF protein may be a suitable target for drug developmentagainst breast cancer and a good diagnostic marker for the malignancy ofbreast tumor. SEQ ID NO:1 and polynucleotides comprising this sequenceare therefore useful as hsOAFs. Reference to hsOAF nucleotide or aminoacid sequences includes variants which have similar expression patternsin high metastatic relative to non-metastatic or low metastatic cells.HsOAF polypeptides can differ in length from full-length hsOAF proteinsand contain at least 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220,240, 260, 265, 270 or 271 or more contiguous amino acids of a hsOAFprotein. Exemplary polynucleotides include those encoding amino acidsfrom about 1 to about 273; from 1 to 273; from about 2 to about 273;from 2 to 273; from about 26 to about 273; and from 26 to 273 of SEQ IDNO:2.

Variants of marker proteins and polypeptides can also occur. HsOAFprotein or polypeptide variants can be naturally or non-naturallyoccurring. Naturally occurring hsOAF protein or polypeptide variants arefound in humans or other species and comprise amino acid sequences whichare substantially identical to a protein encoded by a gene correspondingto the nucleotide sequence shown in SEQ ID NO:1 or its complement.Non-naturally occurring hsOAF protein or polypeptide variants whichretain substantially the same differential expression patterns in highmetastatic relative to low-metastatic or non-metastatic breast cancercells as naturally occurring hsOAF protein or polypeptide variants arealso included here. Preferably, naturally or non-naturally occurringhsOAF protein or polypeptide variants have amino acid sequences whichare at least 85%, 90%, 91%, 92%, 93%, 94%, or 95% identical to aminoacid sequences encoded by the nucleotide sequence shown in SEQ ID NO:1.More preferably, the molecules are at least 96%, 97%, 98% or 99%identical. Percent sequence identity between a wild-type protein orpolypeptide and a variant is determined by aligning the wild-typeprotein or polypeptide with the variant to obtain the greatest number ofamino acid matches, as is known in the art, counting the number of aminoacid matches between the wild-type and the variant, and dividing thetotal number of matches by the total number of amino acid residues ofthe wild-type sequence.

Preferably, amino acid changes in hsOAF protein or polypeptide variantsare conservative amino acid changes, i.e., substitutions of similarlycharged or uncharged amino acids. A conservative amino acid changeinvolves substitution of one of a family of amino acids which arerelated in their side chains. Naturally occurring amino acids aregenerally divided into four families: acidic (aspartate, glutamate),basic (lysine, arginine, histidine), non-polar (alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),and uncharged polar (glycine, asparagine, glutamine, cystine, serine,threonine, tyrosine) amino acids. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids.

It is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid will not have a major effect on thebiological properties of the resulting hsOAF protein or polypeptidevariant. Properties and functions of hsOAF protein or polypeptidevariants are of the same type as a hsOAF protein or polypeptidecomprising amino acid sequences encoded by the nucleotide sequence shownin SEQ ID NO:1, although the properties and functions of variants candiffer in degree. Whether an amino acid change results in a hsOAFprotein or polypeptide variant with the appropriate differentialexpression pattern can readily be determined. For example, nucleotideprobes can be selected from the marker gene sequences disclosed hereinand used to detect marker gene mRNA in Northern blots or in tissuesections, as is known in the art. Alternatively, antibodies whichspecifically bind to protein products of hsOAF genes can be used todetect expression of hsOAF proteins or variants thereof.

HsOAF variants include glycosylated forms, aggregative conjugates withother molecules, and covalent conjugates with unrelated chemicalmoieties. HsOAF variants also include allelic variants, speciesvariants, and muteins. Truncations or deletions of regions which do notaffect the differential expression of hsOAF genes are also hsOAFvariants. Covalent variants can be prepared by linking functionalitiesto groups which are found in the amino acid chain or at the N- orC-terminal residue, as is known in the art.

It will be recognized in the art that some amino acid sequence of thepolypeptide of the invention can be varied without significant effect onthe structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there arecritical areas on the protein which determine activity. In general, itis possible to replace residues that form the tertiary structure,provided that residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein. Thereplacement of amino acids can also change the selectivity of binding tocell surface receptors. Ostade et al., Nature 361:266–268 (1993)describes certain mutations resulting in selective binding of TNF-alphato only one of the two known types of TNF receptors. Thus, thepolypeptides of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

The invention further includes variations of the disclosed polypeptidewhich show comparable expression patterns or which include antigenicregions. Such mutants include deletions, insertions, inversions,repeats, and type substitutions. Guidance concerning which amino acidchanges are likely to be phenotypically silent can be found in Bowie, J.U., et al., “Deciphering the Message in Protein Sequences: Tolerance toAmino Acid Substitutions,” Science 247:1306–1310 (1990).

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the disclosed protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331–340 (1967); Robbins et al., Diabetes36:838–845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug CarrierSystems 10:307–377 (1993)).

Amino acids in the polypeptides of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081–1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding, or in vitro proliferative activity.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899–904 (1992) and de Vos et al. Science 255:306–312 (1992)).

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein. Of course, the number of aminoacid substitutions a skilled artisan would make depends on many factors,including those described above. Generally speaking, the number ofsubstitutions for any given polypeptide will not be more than 50, 40,30, 25, 20, 15, 10, 5 or 3.

Full-length hsOAF proteins can be extracted, using standard biochemicalmethods, from hsOAF protein-producing human cells, such as metastaticbreast cancer cells. An isolated and purified hsOAF protein orpolypeptide is separated from other compounds which normally associatewith a hsOAF protein or polypeptide in a cell, such as certain proteins,carbohydrates, lipids, or subcellular organelles. A preparation ofisolated and purified hsOAF proteins or polypeptides is at least 80%pure; preferably, the preparations are 90%, 95%, or 99% pure.

A human gene encoding SEQ ID NO:2 can be identified and isolated usingmethods know in the art. According to one method, SEQ ID NO:1 isprepared in a computer-readable format. The sequence is compared withpolynucleotide sequences of a human genome, and one or more human genomesequences having at least 95% sequence identity to SEQ ID NO:1 areidentified, for example by using the Smith-Waterman algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 1 as parameters. Probes based on the regions of homologybetween SEQ ID NO:1 and the human genome sequences are prepared and usedto isolate polynucleotides from human genomic DNA, using methods knownin the art. As of the filing date a human polynucleotide correspondingto the full polynucleotide of SEQ ID NO:1 was not identified in thepublic databases. Thus, the invention includes human genomicDNAcomprising the coding region of SEQ ID NO:1 and any untranslatedregions which do not share homology with SEQ ID NO:1 but which arecontiguous with homologous regions. Such genomic DNA includes but is notlimited to introns, promoters, and other regulatory regions functionallyassociated with a human gene having a region encoding SEQ ID NO:2.

hsOAF proteins and polypeptides can also be produced by recombinant DNAmethods or by synthetic chemical methods. For production of recombinanthsOAF proteins or polypeptides, coding sequences selected from thenucleotide sequences shown in SEQ ID NO:1, or variants of thosesequences which encode hsOAF proteins, can be expressed in knownprokaryotic or eukaryotic expression systems (see below). Bacterial,yeast, insect, or mammalian expression systems can be used, as is knownin the art.

Alternatively, synthetic chemical methods, such as solid phase peptidesynthesis, can be used to synthesize a hsOAF protein or polypeptide.General means for the production of peptides, analogs or derivatives areoutlined in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, ANDPROTEINS—A SURVEY OF RECENT DEVELOPMENTS, Weinstein, B. ed., MarcellDekker, Inc., publ., New York (1983). Moreover, substitution of D-aminoacids for the normal L-stereoisomer can be carried out to increase thehalf-life of the molecule. hsOAF variants can be similarly produced.

Non-naturally occurring fusion proteins comprising at least 6, 8, 10,12, 15, 18, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 265, 270 or 271 ormore contiguous hsOAF amino acids can also be constructed. Human hsOAFfusion proteins are useful for generating antibodies against hsOAF aminoacid sequences and for use in various assay systems. For example, hsOAFfusion proteins can be used to identify proteins which interact withhsOAF proteins and influence their functions. Physical methods, such asprotein affinity chromatography, or library-based assays forprotein-protein interactions, such as the yeast two-hybrid or phagedisplay systems, can also be used for this purpose. Such methods arewell known in the art and can also be used as drug screens.

A hsOAF fusion protein comprises two protein segments fused together bymeans of a peptide bond. The first protein segment comprises at least 6,8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 265, 270 or271 or more contiguous amino acids of a hsOAF protein. The amino acidscan be selected from the amino acid sequences encoded by the nucleotidesequence shown in SEQ ID NO:1 or from variants of the sequence, such asthose described above. The first protein segment can also comprise afull-length hsOAF protein.

In one preferred embodiment, the first protein segment comprises thepolypeptide shown in SEQ ID NO:2. In avariation of this embodiment, thefirst protein segment consists of amino acids 31–287 of SEQ ID NO:2.This fusion protein lacks the signal peptide of SEQ ID NO:2 and would besuitable for retention of the expressed fusion protein inside the cell.

The second protein segment can be a full-length protein or a proteinfragment or polypeptide not found adjacent to the first protein segmentin the native protein encoded by SEQ ID NO:1. The fusion protein can belabeled with a detectable marker, as is known in the art, such as aradioactive, fluorescent, chemiluminescent, or biotinylated marker. Thesecond protein segment can be an enzyme which will generate a detectableproduct, such as β-galactosidase. The first protein segment can beN-terminal or C-terminal, as is convenient.

Techniques for making fusion proteins, either recombinantly or bycovalently linking two protein segments, are also well known.Recombinant DNA methods can be used to prepare hsOAF fusion proteins,for example, by making a DNA construct which comprises coding sequencesof SEQ ID NO:1 in proper reading frame with nucleotides encoding thesecond protein segment and expressing the DNA construct in a host cell,as described below. The open reading frame of SEQ ID NO:1 is shown inFIG. 4.

Isolated and purified hsOAF proteins, polypeptides, variants, or fusionproteins can be used as immunogens, to obtain preparations of antibodieswhich specifically bind to a hsOAF protein. The antibodies can be used,interalia, to detect wild-type hsOAF proteins in human tissue andfractions thereof. The antibodies can also be used to detect thepresence of mutations in hsOAF genes which result in under- orover-expression of a hsOAF protein or in expression of a hsOAF proteinwith altered size or electrophoretic mobility.

Preparations of polyclonal or monoclonal antibodies can be made usingstandard methods. Single-chain antibodies can also be prepared. Apreferred immunogen is a polypeptide comprising SEQ ID NO:2.Single-chain antibodies which specifically bind to hsOAF proteins,polypeptides, variants, or fusion proteins can be isolated, for example,from single-chain immunoglobulin display libraries, as is known in theart. The library is “panned” against hsOAF protein amino acid sequencesof SEQ ID NO:2, and a number of single chain antibodies which bind withhigh-affinity to different epitopes of hsOAF proteins can be isolated.Hayashi et al., 1995, Gene 160:129–30. Single-chain antibodies can alsobe constructed using a DNA amplification method, such as the polymerasechain reaction (PCR), using hybridoma cDNA as a template. Thirion etal., 1996, Eur. J. Cancer Prev. 5:507–11.

HsOAF-specific antibodies specifically bind to epitopes present in afull-length hsOAF protein having an amino acid sequence encoded by anucleotide sequence shown in SEQ ID NO:1, to hsOAF polypeptides, or tohsOAF variants, either alone or as part of a fusion protein. Preferably,hsOAF epitopes are not present in other human proteins. Typically, atleast 6, 8, 10, or 12 contiguous amino acids are required to form anepitope. However, epitopes which involve non-contiguous amino acids mayrequire more, e.g., at least 15, 25, or 50 amino acids.

Antibodies which specifically bind to hsOAF proteins, polypeptides,fusion proteins, or variants provide a detection signal at least 5-,10-, or 20-fold higher than a detection signal provided with otherproteins when used in Western blots or other immunochemical assays.Preferably, antibodies which specifically bind to hsOAF epitopes do notdetect other proteins in immunochemical assays and can immunoprecipitatea hsOAF protein, polypeptide, fusion protein, or variant from solution.In a preferred method, hsOAF protein expression is detected using animmunohistochemical staining kit, such as that of BioGenex Laboratories,Inc. (San Ramon, Calif.).

Subgenomic polynucleotides contain less than a whole chromosome.Preferably, the polynucleotides are intron-free. In a preferredembodiment, the polynucleotide molecules comprise a contiguous sequenceof 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300 or 2350 nucleotides from SEQ ID NO:1 or thecomplements thereof. The complement of a nucleotide sequence shown inSEQ ID NO:1 is a contiguous nucleotide sequence which forms Watson-Crickbase pairs with a contiguous nucleotide sequence shown in SEQ ID NO:1.

Degenerate nucleotide sequences encoding amino acid sequences of hsOAFprotein or variants, as well as homologous nucleotide sequences whichcomprise a polynucleotide at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the coding region of the nucleotidesequence shown in SEQ ID NO:1, are also hsOAF subgenomicpolynucleotides. Typically, homologous hsOAF subgenomic polynucleotidesequences can be confirmed by hybridization under stringent conditions,as is known in the art. Percent sequence identity between wild-type andhomologous variant sequences is determined by aligning the wild-typepolynucleotide with the variant to obtain the greatest number ofnucleotide matches, as is known in the art, counting the number ofnucleotide matches between the wild-type and the variant, and dividingthe total number of matches by the total number of nucleotides of thewild-type sequence. A preferred algorithm for calculating percentidentity is the Smith-Waterman homology search algorithm as implementedin MPSRCH program (Oxford Molecular) using an affine gap search with thefollowing search parameters: gap open penalty of 12, and gap extensionpenalty of 1.

A hsOAF subgenomic polynucleotide comprising hsOAF protein codingsequences can be used in an expression construct. Preferably, the hsOAFsubgenomic polynucleotide is inserted into an expression plasmid (forexample, the Ecdyson system, pIND, In vitro Gene). HsOAF subgenomicpolynucleotides can be propagated in vectors and cell lines usingtechniques well known in the art. HsOAF subgenomic polynucleotides canbe on linear or circular molecules. They can be on autonomouslyreplicating molecules or on molecules without replication sequences.They can be regulated by their own or by other regulatory sequences, asare known in the art.

A host cell comprising a hsOAF expression construct can then be used toexpress all or a portion of a hsOAF protein. Host cells comprising hsOAFexpression constructs can be prokaryotic or eukaryotic. A variety ofhost cells are available for use in bacterial, yeast, insect, and humanexpression systems and can be used to express or to propagate hsOAFexpression constructs (see below). Expression constructs can beintroduced into host cells using any technique known in the art. Thesetechniques include transferrin-polycation-mediated DNA transfer,transfection with naked or encapsulated nucleic acids, liposome-mediatedcellular fusion, intracellular transportation of DNA-coated latex beads,protoplast fusion, viral infection, electroporation, and calciumphosphate-mediated transfection.

A hsOAF expression construct comprises a promoter which is functional ina chosen host cell. The skilled artisan can readily select anappropriate promoter from the large number of cell type-specificpromoters known and used in the art. The expression construct can alsocontain a transcription terminator which is functional in the host cell.The expression construct comprises a polynucleotide segment whichencodes all or a portion of the hsOAF protein, variant, fusion protein,antibody, or ribozyme. The polynucleotide segment is located downstreamfrom the promoter. Transcription of the polynucleotide segment initiatesat the promoter. The expression construct can be linear or circular andcan contain sequences, if desired, for autonomous replication.

Bacterial systems for expressing hsOAF expression constructs includethose described in Chang et al., Nature (1978) 275:615, Goeddel et al.,Nature (1979) 281:544, Goeddel et al., Nucleic Acids Res. (1980) 8:4057,EP 36,776, U.S. Pat. No. 4,551,433, deBoer et al., Proc. Nat'l Acad.Sci. USA (1983) 80:21–25, and Siebenlist et al., Cell (1980) 20:269.

Expression systems in yeast include those described in Hinnen et al.,Proc. Nat'l Acad. Sci. USA (1978) 75:1929; Ito et al., J. Bacteriol.(1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze etal., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459, Roggenkamp et al., Mol. Gen. Genet. (1986)202:302) Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737, Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376, U.S. Pat. Nos. 4,837,148,4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380, Gaillardin et al., Curr. Genet. (1985) 10:49,Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284–289;Tilburn et al., Gene (1983) 26:205–221, Yelton et al., Proc. Nat'l Acad.Sci. USA (1984) 81:1470–1474, Kelly and Hynes, EMBO J. (1985) 4:475479;EP 244,234, and WO 91/00357.

Expression of hsOAF expression constructs in insects can be carried outas described in U.S. Pat. No. 4,745,051, Friesen et al. (1986) “TheRegulation of Baculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OFBACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP 155,476, and Vlak etal., J. Gen. Virol. (1988) 69:765–776, Miller et al., Ann. Rev.Microbiol. (1988) 42:177, Carbonell et al., Gene (1988) 73:409, Maeda etal., Nature (1985) 315:592–594, Lebacq-Verheyden et al., Mol. Cell.Biol. (1988) 8:3129; Smith et al., Proc. Nat'l Acad. Sci. USA (1985)82:8404, Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA(1988) 7:99. Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts are described in Luckow et al.,Bio/Technology (1988) 6:47–55, Miller et al., in GENETIC ENGINEERING(Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing, 1986),pp.277–279, and Maeda et al., Nature, (1985) 315:592–594.

Mammalian expression of hsOAF expression constructs can be achieved asdescribed in Dijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc.Nat'l Acad. Sci. USA (1982b) 79:6777, Boshart et al., Cell (1985) 41:521and U.S. Pat. No. 4,399,216. Other features of mammalian expression ofhsOAF expression constructs can be facilitated as described in Ham andWallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980)102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO90/103430, WO 87/00195, and U.S. RE 30,985.

Subgenomic polynucleotides of the invention can also be used in genedelivery vehicles, for the purpose of delivering a hsOAF mRNA oroligonucleotide (either with the sequence of native hsOAF mRNA or itscomplement), full-length hsOAF protein, hsOAF fusion protein, hsOAFpolypeptide, or hsOAF-specific ribozyme or single-chain antibody, into acell, preferably a eukaryotic cell. According to the present invention,a gene delivery vehicle can be, for example, naked plasmid DNA, a viralexpression vector comprising a hsOAF subgenomic polynucleotide, or ahsOAF subgenomic polynucleotide in conjunction with a liposome or acondensing agent.

The invention provides a method of detecting hsOAF gene expression in abiological sample. Detection of hsOAF gene expression is useful, forexample, for identifying metastases or for determining metastaticpotential in a tissue sample, preferably a tumor. Appropriate treatmentregimens can then be designed for patients who are at risk fordeveloping metastatic cancers in other organs of the body.

The body sample can be, for example, a solid tissue or a fluid sample.The native polypeptide encoded by SEQ ID NO:1 is a putative secretedprotein, and is detected in body fluids including blood and lymphaticfluid, particularly those draining from tumor sites in the body. Proteinor nucleic acid expression products can be detected in the body sample.In one embodiment, the body sample is assayed for the presence of ahsOAF protein. A hsOAF protein comprises a sequence encoded by anucleotide sequence shown in SEQ ID NO:1 or its complement and can bedetected using the hsOAF protein-specific antibodies of the presentinvention. The antibodies can be labeled, for example, with aradioactive, fluorescent, biotinylated, or enzymatic tag and detecteddirectly, or can be detected using indirect immunochemical methods,using a labeled secondary antibody. The presence of the hsOAF proteinscan be assayed, for example, in tissue sections by immunocytochemistry,or in lysates, using Western blotting, as is known in the art.

In another embodiment, the body sample is assayed for the presence ofmarker protein mRNA. A sample can be contacted with a nucleic acidhybridization probe capable of hybridizing with the mRNA correspondingthe selected polypeptide. Still further, the sample can be subjected toa Northern blotting technique to detect mRNA, indicating expression ofthe polypeptide. For those techniques in which mRNA is detected, thesample can be subjected to a nucleic acid amplification process wherebythe mRNA molecule or a selected part thereof is amplified usingappropriate nucleotide primers. Other RNA detection techniques can alsobe used, including, but not limited to, in situ hybridization.

Marker protein-specific probes can be generated using the cDNA sequencedisclosed in SEQ ID NO:1. The probes are preferably at least 15 to 50nucleotides in length, although they can be at least 8, 10, 11, 12, 20,25, 30, 35, 40, 45, 60, 75, or 100 or more nucleotides in length. Apreferable region for selecting probes is within nucleotide positions446–1173 of SEQ ID NO:1. The probes can be synthesized chemically or canbe generated from longer polynucleotides using restriction enzymes. Theprobes can be labeled, for example, with a radioactive, biotinylated, orfluorescent tag.

Optionally, the level of a particular hsOAF expression product in a bodysample can be quantitated. Quantitation can be accomplished, forexample, by comparing the level of expression product detected in thebody sample with the amounts of product present in a standard curve. Acomparison can be made visually or using a technique such asdensitometry, with or without computerized assistance. For use ascontrols, body samples can be isolated from other humans, othernon-cancerous organs of the patient being tested, or non-metastaticbreast cancer from the patient being tested. As indicated by the resultsherein, expression of SEQ ID NO:1 in low-metastatic or non-metastaticbreast cancer cells is between 3% and 44% of the expression levels inhighly-metastatic breast cancer cells. If expression in a test sample isat least 2-fold greater than in a suitable control sample, this isindicative of metastatic cells.

Polynucleotides encoding hsOAF-specific reagents of the invention, suchas antibodies and nucleotide probes, can be supplied in a kit fordetecting marker gene expression products in a biological sample. Thekit can also contain buffers or labeling components, as well asinstructions for using the reagents to detect the marker expressionproducts in the biological sample.

Expression of a hsOAF gene can be altered using an antisenseoligonucleotide sequence. The antisense sequence is complementary to atleast a portion of the coding sequence (nucleotides 365–1173) of a hsOAFgene having a nucleotide sequence shown in SEQ ID NO:1. Preferably, theantisense oligonucleotide sequence is at least six nucleotides inlength, but can be at least about 8, 12, 15, 20, 25, 30, 35, 40, 45, or50 nucleotides long. Longer sequences can also be used. Antisenseoligonucleotide molecules can be provided in a DNA construct andintroduced into cells whose division is to be decreased. Such cellsinclude highly-metastatic breast cancer cells.

Antisense oligonucleotides can comprise deoxyribonucleotides,ribonucleotides, or a combination of both. Oligonucleotides can besynthesized manually or by an automated synthesizer, by covalentlylinking the 5′ end of one nucleotide with the 3′ end of anothernucleotide with non-phosphodiester internucleotide linkages suchalkylphosphonates, phosphorothioates, phosphorodithioates,alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphateesters, carbamates, acetamidate, carboxymethyl esters, carbonates, andphosphate triesters. See Brown, 1994, Meth. Mol. Biol. 20:1–8; Sonveaux,1994, Meth. Mol. Biol. 26:1–72; Uhlmann et al., 1990, Chem. Rev.90:543–583.

Antibodies of the invention which specifically bind to a hsOAF proteincan also be used to alter hsOAF gene expression. By antibodies is meantantibodies and parts or derivatives thereof, such as single chainantibodies, that retain specific binding for the protein. Specificantibodies bind to hsOAF proteins and prevent the proteins fromfunctioning in the cell. Polynucleotides encoding specific antibodies ofthe invention can be introduced into cells, as described above.

Marker proteins of the present invention can be used to screen for drugswhich have a therapeutic anti-metastatic effect. The effect of a testcompound on hsOAF protein synthesis can also be used to identify testcompounds which modulate metastasis. Test compounds which can bescreened include any substances, whether natural products or synthetic,which can be administered to the subject. Libraries or mixtures ofcompounds can be tested. The compounds or substances can be those forwhich a pharmaceutical effect is previously known or unknown.

Synthesis of hsOAF proteins can be measured by any means for measuringprotein synthesis known in the art, such as incorporation of labeledamino acids into proteins and detection of labeled hsOAF proteins in apolyacrylamide gel. The amount of hsOAF proteins can be detected, forexample, using hsOAF protein-specific antibodies of the invention inWestern blots. The amount of the hsOAF proteins synthesized in thepresence or absence of a test compound can be determined by any meansknown in the art, such as comparison of the amount of hsOAF proteinsynthesized with the amount of the hsOAF proteins present in a standardcurve.

The effect of a test compound on hsOAF protein synthesis can also bemeasured by Northern blot analysis, by measuring the amount of hsOAFprotein mRNA expression in response to the test compound using hsOAFprotein specific nucleotide probes of the invention, as is known in theart.

Typically, a biological sample is contacted with a range ofconcentrations of the test compound, such as 1.0 nM, 5.0 nM, 10 nM, 50nM, 100 nM, 500 nM, 1 mM, 10 mM, 50 mM, and 100 mM. Preferably, the testcompound decreases expression of a hsOAF protein by 60%, 75%, or 80%.More preferably, a decrease of 85%, 90%, 95%, or 98% is achieved.

The invention provides compositions for decreasing expression of hsOAFprotein. These compositions comprise polynucleotides encoding all or atleast a portion of a hsOAF protein gene expression product. Preferably,the therapeutic composition contains an expression construct comprisinga promoter and a polynucleotide segment encoding at least a portion ofthe hsOAF protein which is effective to decrease metastatic potential.Portions of hsOAF genes or proteins which are effective to decreasemetastatic potential of a cell can be determined, for example, byintroducing portions of hsOAF genes or polypeptides into metastatic celllines, such as MDA-MB-231, MDA-MB-435, Km12C, or Km12L4, and assayingthe division rate of the cells or the ability of the cells to formmetastases when implanted in vivo, as is known in the art.Non-metastatic cell lines can be used to assay the ability of a portionof a hsOAF protein to increase expression of a hsOAF gene.

Typically, a therapeutic hsOAF composition is prepared as an injectable,either as a liquid solution or suspension; however, solid forms suitablefor solution in, or suspension in, liquid vehicles prior to injectioncan also be prepared. A hsOAF composition can also be formulated into anenteric coated tablet or gel capsule according to known methods in theart, such as those described in U.S. Pat. No. 4,853,230, EP 225,189, AU9,224,296, and AU 9,230,801.

Administration of the hsOAF therapeutic agents of the invention caninclude local or systemic administration, including injection, oraladministration, particle gun, or catheterized administration, andtopical administration. Various methods can be used to administer atherapeutic hsOAF composition directly to a specific site in the body.

For treatment of tumors, including metastatic lesions, for example, atherapeutic hsOAF composition can be injected several times in severaldifferent locations within the body of tumor. Alternatively, arterieswhich serve a tumor can be identified, and a therapeutic compositioninjected into such an artery, in order to deliver the compositiondirectly into the tumor.

A tumor which has a necrotic center can be aspirated and the compositioninjected directly into the now empty center of the tumor. A therapeutichsOAF composition can be directly administered to the surface of atumor, for example, by topical application of the composition. X-rayimaging can be used to assist in certain of the above delivery methods.Combination therapeutic agents, including a hsOAF proteins orpolypeptide or a hsOAF subgenomic polynucleotide and other therapeuticagents, can be administered simultaneously or sequentially.

Alternatively, a hsOAF therapeutic composition can be introduced intohuman cells ex vivo, and the cells then replaced into the human. Cellscan be removed from a variety of locations including, for example, froma selected tumor or from an affected organ. In addition, a therapeuticcomposition can be inserted into non-affected cells, for example, dermalfibroblasts or peripheral blood leukocytes. If desired, particularfractions of cells such as a T cell subset or stem cells can also bespecifically removed from the blood (see, for example, PCT WO 91/16116).The removed cells can then be contacted with a hsOAF therapeuticcomposition utilizing any of the above-described techniques, followed bythe return of the cells to the human, preferably to or within thevicinity of a tumor or other site to be treated. The methods describedabove can additionally comprise the steps of depleting fibroblasts orother non-contaminating tumor cells subsequent to removing tumor cellsfrom a human, and/or the step of inactivating the cells, for example, byirradiation.

Both the dose of a therapeutic composition and the means ofadministration can be determined based on the specific qualities of thetherapeutic composition, the condition, age, and weight of the patient,the progression of the disease, and other relevant factors. Preferably,a therapeutic composition of the invention decreases expression of thehsOAF genes by 50%, 60%, 70%, or 80%. Most preferably, expression of thehsOAF genes is decreased by 90%, 95%, 99%, or 100%. The effectiveness ofthe mechanism chosen to alter expression of the hsOAF genes can beassessed using methods well known in the art, such as hybridization ofnucleotide probes to mRNA of the hsOAF genes, quantitative RT-PCR, ordetection of the hsOAF proteins using specific antibodies of theinvention.

If the composition contains the hsOAF proteins, polypeptide, orantibody, effective dosages of the composition are in the range of about5 μg to about 50 μg/kg of patient body weight, about 50 μg to about 5mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about200 to about 250 μg/kg.

Therapeutic compositions containing hsOAF subgenomic polynucleotides,such as antisense oligonucleotides, can be administered in a range ofabout 100 ng to about 200 mg of DNA for local administration.Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNAcan also be used during a gene therapy protocol. Factors such as methodof action and efficacy of transformation and expression areconsiderations that will affect the dosage required for ultimateefficacy of the hsOAF subgenomic polynucleotides. Where greaterexpression is desired over a larger area of tissue, larger amounts ofhsOAF subgenomic polynucleotides or the same amounts readministered in asuccessive protocol of administrations, or several administrations todifferent adjacent or close tissue portions of, for example, a tumorsite, can be required to effect a positive therapeutic outcome. In allcases, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect.

Expression of an endogenous hsOAF gene in a cell can also be altered byintroducing in frame with the endogenous hsOAF gene a DNA constructcomprising a hsOAF protein targeting sequence, a regulatory sequence, anexon, and an unpaired splice donor site by homologous recombination,such that a homologously recombinant cell comprising the DNA constructis formed. The new transcription unit can be used to turn the hsOAF geneon or off as desired. This method of affecting endogenous geneexpression is taught in U.S. Pat. No. 5,641,670, which is incorporatedherein by reference.

A hsOAF subgenomic polynucleotide can also be delivered to subjects forthe purpose of screening test compounds for those which are useful forenhancing transfer of hsOAF subgenomic polynucleotides to the cell orfor enhancing subsequent biological effects of hsOAF subgenomicpolynucleotides within the cell. Such biological effects includehybridization to complementary hsOAF mRNA and inhibition of itstranslation, expression of a hsOAF subgenomic polynucleotide to formhsOAF mRNA and/or hsOAF protein, and replication and integration of ahsOAF subgenomic polynucleotide. The subject can be a cell culture or ananimal, preferably a mammal, more preferably a human.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLES Materials and Methods

Human tissues. Normal human tissues were obtained as Human Total RNAPanels, Clontech. Tissue samples were also obtained from breast cancerpatients, and included primary breast tumors and metastases.

Cell culture. MDA-MB-435, MDA-MB-231, ALAB, MDA-MB-468, MDA-MB-361,ZR-75-1, MCF-7, MDA-MB-453 and SK-BR-3 human breast cancer cell lines(obtained from Chiron Master Culture Collection, Chiron Corporation)were grown at 37° C. in 5% CO₂ in DMEM+ HAM'S F-12 (1:1) (Bio*Whittaker,Walkersville, Md.) containing 2 mM L-Glutamine, 1 mM Sodium Pyruvate,100 U/ml Penicillin and 100 μg/ml Streptomycin (Bio*Whittaker,Walkersville, Md.), 1× Vitamin Solution, 1×Non-Essential Amino Acids(Irvine Scientific, Santa Ana, Calif.), and 10% heat-inactivated fetalbovine serum (Life Technologies, Rockville, Md.). COS-7 cells wereobtained from ATCC and grown at 37° C. in 5% CO₂ in DMEM with 10%heat-inactivated fetal bovine serum (Life Technologies).

Concentration of Opti-MEM1 supernatant. Opti-MEM1 (Life Technologies)culture media were concentrated through Centricon YM-10 and/or MicroconYM-10 columns (Millipore Corporation, Bedford, Mass.). SDS-PAGE sampleloading buffer was then added and the samples were boiled.

Northern blot hybridization. Total RNAs were prepared from culturedbreast cancer cell lines and tumor tissues of SCID mice transplantedwith breast cancer cell lines with RNeasy Maxi Kit (Qiagen, Valencia,Calif.). Approximately 20 μg of total RNA per lane was loaded onto aformaldehyde/agarose gel for electrophoresis, then transferred to aHybond-N+ nylon membrane (Amersham Life Science, Little Chalfont,England). The blot was fixed by UV irradiation. Rapid-Hyb buffer(Amersham Life Science) with 5 mg/ml denatured single stranded sperm DNAwas pre-warmed to 65° C. and the blot was pre-hybridized in the bufferwith shaking at 65° C. for 30 minutes. A hsOAF cDNA fragment or aβ-actin cDNA fragment as probe labeled with [α-³²P]dCTP (3000 Ci/mmol,Amersham Pharmacia Biotech Inc., Piscataway, N.J.) (Prime-It RmT Kit,Stratagene, La Jolla, Calif.) and purified with ProbeQuant™ G-50 MicroColumn (Amersham Pharmacia Biotech Inc.) was added and hybridized to theblot with shaking at 65° C. for overnight. The blot was washed in 2×SSC,0.1%(w/v) SDS at room temperature for 20 minutes, twice in 1×SSC, 0.1%(w/v) SDS at 65° C. for 15 minutes, then exposed to Hyperfilms (AmershamLife Science).

Immunoblotting. Protein samples were subjected to electrophoresis on10–20% SDS-PAGE gels then transferred to PVDF membranes (0.2 μm) byelectroblotting in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH8.3. Membranes were blocked in TBST (pH 7.5) containing 10% non-fatmilk, then blotted in PBS (pH 7.4) containing 1% BSA with a rabbitanti-hsOAF serum (1:1000), followed by probing with a secondary antibodyalkaline phosphatase-conjugated goat anti-rabbit IgG (1:2000) (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.). Protein bands were thenvisualized by NBT/BCIP reagent (Boehringer Mannheim, Germany).

Transient transfection. The coding region (356–1174) of hsOAF cDNA wascloned into a modified expression vector pRetro-On (Clontech, Palo Alto,Calif.). The pRetro-On vector harboring hsOAF or the control pRetro-Onvector with GFP was transfected into COS-7 cells on a 100 mm cultureplate using Effectene™ Transfection Reagent Kit (Qiagen) as instructedin the protocol provided by the manufacturer. Cells were recovered inDMEM with 10% FBS for overnight then switched to Opti-MEM1. After twomore days, the supernatant was collected and concentrated for westernblot analysis.

Antisense oligo transfection. MDA-MB-435 cells were seeded on 6-wellculture plates one day before transfection to yield a 90% density attransfection. 100 μM antisense or reverse control oligo was diluted to 2μM in Opti-MEM1 for transfection. 0.5 mM sterile lipitoid1 was dilutedto a ratio of 1.5 nmol lipitoid1: 1 μg oligo in the same volume ofOpti-MEM1. The diluted oligo and the diluted lipitoid1 were mixed andimmediately added to cells in culture media to a final concentration of100, 200, or 300 nM oligo. After 6 hrs, the transfection mixture wasreplaced with normal culture media and cells were incubated for recoveryfor overnight. The sequence of the antisense oligo isAGCTGCGGATGCCACACTTGTAGG (SEQ ID NO:4) and the sequence of the reversecontrol oligo is GGATGTTCACACCGTAGGCGTCGA (SEQ ID NO:5).

Matrigel invasion assay. Cells were trypsinized, washed, and resuspendedin media for counting. 4×10⁴ cells were washed and resuspended in 100 μlmedia on ice. 200 μl Matrigel (Collaborative Biomedical Products,Bedford, Mass.) was added to the cells on ice. The Matrigel and thecells were carefully mixed then dispensed into a well of 24-well cultureplate and solidified at 37° C. for 30 min. The Matrigel-cell mixture wastopped with 0.5 ml medium and incubated at 37° C. in 5% CO₂ for 6 days.The medium was replenished every 2 days.

Proliferation assay. Cells were trypsinized, washed, and resuspended inmedia for counting. Cells were then transferred into 96-well plates(5000 cells/well) for incubation. Cell numbers were measured withQuantos™ Cell Proliferation Assay Kit (Stratagene, La Jolla, Calif.)every day.

Preparation of hsOAF polyclonal antibody. hsOAF antisera were generatedin two rabbits immunized against the C-terminal peptide (H-FYVPQRQLCLWDEDPYPG-OHN, KLH conjugated, SEQ ID NO: 11), and then affinitypurification was conducted to obtain the hsOAF polyclonal antibody(ResGen, an Invitrogen Corporation, Huntsville, Ala.). The antibodypreparation was titrated by ELISA assay.

Immunohistochemical staining. Immunohistochemical staining was performedto detect hsOAF protein expression in tissues with the hsOAF polyclonalantibody using the immunohistochemical staining kits from BioGenexLaboratories, Inc. (San Ramon, Calif.). All procedures were carried outas instructed in the protocol provided by the manufacturer.

Example 1 Identification of a Human cDNA Sequence

DNA encoding a putative human homologue of the Drosophila Out at First(oaf) gene is shown in SEQ ID NO:1. An alignment of hsOAF andDrosophilia OAF is shown in FIG. 7. The polynucleotide comprises 2366base pairs, and an open reading frame is identified. A translation ofthe ORF, a polypeptide of 273 amino acids, is shown in SEQ ID NO:2. FIG.4 provides the DNA and amino acid sequences, indicating the position ofthe ORF. The first 30 amino acids form a signal peptide, indicating thatthe protein may be secreted. The amino acid sequence of the signalpeptide is: MRLPGVPLARPALLLLLPLLAPLLG#TGAPA (SEQ ID NO:3). “#” indicatesthe location of the predicted protease cut site.

Example 2 Expression of hsOAF in Primary and Metastatic Breast CancerTissue

To further understand the importance of hsOAF gene expression in breastcancer, immunohistochemical staining in tissue samples from breastcancer patient was conducted using the hsOAF polyclonal antibody (FIG.5). Strong hsOAF expression was detected not only in all metastases(26/26) but also in almost all primary breast tumors (44/45). Meanwhile,a weak hsOAF positive staining was observed in 8 out of 24 normal breasttissue samples. These results suggest that up-regulation of hsOAF geneexpression may play important roles in both mammary tumor formation anddevelopment.

Example 3 Differential Expression of SEQ ID NO:1 in Breast Cancer CellLines

Expression of SEQ ID NO:1 in the following human breast cancer celllines was compared:

MDA-MB-361, derived from human breast adenocarcinoma;

MDA-MB-231, derived from human breast cancer cells metastatic to boneand/or lung;

MDA-MB-468, derived from estrogen receptor-negative human breast cancercells;

MDA-MB-435, derived from estrogen receptor-negative human breastcarcinoma cells;

MCF-7, derived from non-metastatic human breast cancer cells; and

ZR-75-1, derived from estrogen receptor-positive human breast carcinomacells.

Expression of SEQ ID NO:1 was measured in the highly metastatic breastcancer cell lines MDA-MB 231 and MDA-MB-435, and compared withlow-metastatic or non-metastatic breast cancer cell lines. Expression inMDA-MB-361 was 11% of the level in MDA-MB-231; expression in MDA-MD-468was 44% of the level in MDA-MB-231; expression in MCF-7 was 17% of thelevel in MDA-MB-231; and expression in ZR-75-1 was 12% of the level inMDA-MB-231.

Expression in MDA-MB-361 was 6% of the level in MDA-MB-435; expressionin MDA-MB-468 was 36% of the level in MDA-MB-435; and expression inMCF-7 was 3% of the level in MDA-MB-435. Thus, as shown in Table 2,there is a clear trend of increased expression of SEQ ID NO:1 in breastcancer cell lines derived from human tumors with high metastaticpotential.

TABLE 2 Low Metastatic Cell Lines: High Metastatic % Expression Relativeto High Metastatic Cell Line Cell Line MDA-MB-361 MDA-MB-468 MCF-7ZR-75-1 MDA-MB-231 11% 44% 17% 12% MDA-MB-435  6% 36%  3% ND

A similar expression pattern of this gene remained in tumor tissuesamples from SCID mice transplanted with tumorigenic mammary carcinomacell lines. (FIG. 6.)

Example 4 hsOAF Encodes a Secreted Protein and hsOAF Protein SecretionLevels are Consistent with hsOAF mRNA Expression Levels of MammaryCarcinoma Cell Lines

A predicted signal peptide sequence is located at the N-terminus of thededuced amino acid sequence of hsOAF gene (FIG. 3). To verify thesecretion of hsOAF protein, transient transfection of COS-7 cells andMCF-7 cells was performed with vector pRetro-On harboring hsOAF cDNA.Meanwhile, vector pRetro-On harboring GFP was used as control. Using ahsOAF rabbit antiserum, secreted hsOAF protein was detected in Opti-MEM1culture media of both cell lines after transfection with hsOAF byimmunoblotting (FIG. 8A). Secreted hsOAF protein was probablyglycosylated since multiple bands with higher apparent molecular weightswere seen (the predicted MW of secreted hsOAF protein is 28 Kda). Thesame hsOAF antiserum was used to detect the secretion of hsOAF proteinby various mammary carcinoma cell lines. The secretion levels of hsOAFprotein were consistent with the hsOAF mRNA expression levels amongthese cell lines overall: highly metastatic cell lines showed muchstronger hsOAF secretion than low metastatic/nonmetastatic cell lines(FIG. 8B). MDA-MB-435 had the strongest hsOAF protein secretion.

Example 5 Knockout of hsOAF Expression in MDA-MB-435 Cells by AntisenseOligo Caused Morphological Change, Reduced Cell Invasiveness and SlowerProliferation Rate

To determine if high level of hsOAF gene expression is essential for themetastatic potential of human mammary carcinoma cells, antisense oligotechnology was used to knock out hsOAF expression, then the consequenteffects were observed. MDA-MB-435 was chosen since this highlymetastatic cell line showed the strongest hsOAF protein secretion amongall of the breast cancer cell lines examined. Several pairs of hsOAFantisense (AS) and reverse control (RC) oligos were chosen to test fortheir ability to shut down hsOAF gene expression at the mRNA level.Real-time quantitative RT-PCR analysis in Lightcycler (RocheDiagnostics, Indianapolis, Ind.) was performed to measure hsOAF mRNAlevels in cells. Kang, S. et al., Cancer Research 60(18):5296–5302(2000). The best pair was then selected for the titration of oligoworking concentration. Low oligo concentration is preferred to reducepotential oligo toxicity to cells. The results indicated that treatmentwith 100 nM of the antisense oligo was sufficient to significantlyreduce hsOAF protein secretion of MDA-MB-435 cells. (FIG. 12). This pairof oligos (SEQ ID NO:4 (AS) and 5 (RC)) at 100 nM working concentrationwas used for all the following experiments.

After treatment of MDA-MB-435 cells with hsOAF antisense oligo, dramaticmorphological alteration of cells was observed along with reduced hsOAFprotein secretion (FIG. 10A). Cells became more spherical and lost theirspreading protrusions. Meanwhile, cells treated with reverse controloligo remained similar to the normal tissue cultured MDA-MB-435 cells.Furthermore, culture medium of normal MDA-MB-435 cells containing highlevel of hsOAF protein as the conditioned medium added to cells treatedwith antisense oligo was able to prevent this morphological change,though not completely. This alteration of cell shape may be anindication of reduced invasion ability of cells.

Matrigel invasion assay was then performed to estimate the invasivenessof cells. It has been reported that a stellate, invasive morphology ofbreast cancer cells embedded in matrigel correlates with theirmetastatic potential (Thompson, E. W., et al, J. Cell Physiol.150(3):534–44 (1992); Sugiura, T., et al. J. Cell Biol, 146(6):1375–89(1999); Albini, A., et al., Cancer Res. 47(12):3239–45 (1987); andKramer, R. H., et al., Cancer Res. 46(4 Pt 2):1980–89 (1986)) and thiswas confirmed with various breast cancer cell lines grown in matrigel.Cells were trypsinized, counted, and mixed with matrigel. Media werethen topped on the cell-matrigel mixture. After 6 days of incubation,cell invasion was examined (FIG. 10B). The results showed that cellstreated with hsOAF reverse control oligo formed penetrating, invasive,network-like three-dimensional structures, as the normal MDA-MB-435cells did; on the other hand, cells treated with hsOAF antisense oligoonly formed smooth, spherical colonies. Again, penetrating colonies werealso observed in hsOAF antisense oligo-treated cells incubated in theconditional medium. These data demonstrate that secreted hsOAF proteinis required for the invasiveness and metastatic potential of MDA-MB-435cells.

Additional experiments were performed to examine if secreted hsOAFprotein was involved in MDA-MB-435 cell growth. Cell proliferation assayresults indicated that knockout of hsOAF protein secretion indeed sloweddown proliferation rate of MDA-MB-435 cells, though the change wasmoderate.

Example 6 Northern Blot Analysis of RNA Expression in Human BreastCancer Cell Lines and in Human Tissues

As shown in FIG. 5, mRNA expression was upregulated in metastatic celllines MDA-MB-231 and MDA-MB-435. Total RNA was prepared using the RNeasyKit from Quiagen. Northern blot analysis was performed using 20–30 μgtotal RNA isolated by guanidinium thiocyanate/phenol chloroformextraction from cell lines, from primary tumors, or from metastases inlung. Primary tumors and lung metastasis were developed from cell linesinjected into SCID mice according to methods well known in the art.Plasmids containing partial cDNA clones of hOAF cloned intopCR2.0-TAVector (In vitrogen) were radiolabeled and hybridized at 65° C.in Express-hyb (Clontech). Among all the tissues examined, liver,pancreas, spleen, ovary, and small intestine showed significant hsOAFexpression. HsOAF mRNA expression was also detected in heart, skeletalmuscle, kidney, prostate, colon and bone marrow. (FIG. 9).

Table 3 shows the percentage of hsOAF positives in a variety of tumorsand normal tissues.

TABLE 3 Immunohistochemistry: Percentage of hsOAF positives Tumor NormalPancreas  9/11 0/9 Esophagus 5/8 0/1 Liver 3/6  0/13 Stomach 6/7  6/10Breast 1/1 Hodgkin's 1/8

Example 7 Soft Agar Assay

Soft Agar Assay: The bottom layer consisted of 2 ml of 0.6% agar inmedia plated fresh within a few hours of layering on the cells. For thecell layer, MDA-MB-435 cells as described above were removed from theplate in 0.05% trypsin and washed twice in media. Cells were counted incoulter counter, and resuspended to 106 per ml in media. 10 ml aliquotswere placed with media in 96-well plates (to check counting with WST1),or diluted further for soft agar assay. 2000 cells were diluted in 800ml 0.4% agar in duplicate wells above 0.6% agar bottom layer.

Media layer: After the cell layer agar solidified, 2 ml of media wasbled on top and antisense or reverse control oligo was added withoutdelivery vehicles. Fresh media and oligos were added every 3–4 days.

Colonies were counted in 10 days to 3 weeks. Fields of colonies werecounted by eye. Wst-1 metabolism values were used to compensate forsmall differences in starting cell number. Larger fields can be scannedfor visual record of differences. The results are shown in FIG. 6, inwhich MDA-MB-435 cells treated with antisense formed fewer coloniescompared to cells exposed to the control oligonucleotide.

Those skilled in the art will recognize, or be able to ascertain, usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such specific embodimentsand equivalents are intended to be encompassed by the following claims.

All patents, published patent applications, and publications citedherein are incorporated by reference as if set forth fully herein.

1. An isolated antibody that binds specifically to an isolatedpolypeptide comprising one of the amino acid sequences selected from thegroup consisting of: (a) amino acids from about 1 to about 273 of SEQ IDNO:2; (b) amino acids from about 2 to about 273 of SEQ ID NO:2; and (c)amino acids from 26 to 273 of SEQ ID NO:2.